J. M. McCown

Monoclonal antibodies produced against the four dengue virus serotypes identified four categories of reactions by immunofluorescence: flavivirus group reactive, dengue complex specific, dengue subcomplex specific (DEN-1, DEN-3), and dengue serotype specific. This is the first time that monospecific antibodies have been available for all of these unique antigenic determinants. Hybridoma cell lines producing dengue type-specific antibodies have been deposited in the Hybridoma Cell Bank of the American Type Culture Collection (Rockville, MD) for general distribution.

Monoclonal antibodies directed against antigenic determinants of the New Guinea C strain of dengue-2 virus were obtained from lymphocyte hybridomas produced by fusing immune mouse lymphocytes with mouse myeloma cells. Hybridoma cell culture supernatants were screened by using a radioimmunoassay employing detergent-solubilized dengue-2 infected cell antigens. Monoclonal antibodies in ascitic fluids induced by 22 selected hybridomas were characterized by the hemagglutination-inhibition, plaque reduction neutralization, immunofluorescence, and complement-fixation tests. Both type-specific and broadly cross-reactive antibodies were observed, and immunoglobulin subclasses IgG1 and IgG2a were represented in both groups. At least three distinct antigenic determinants on the virion were defined using these antibodies. A single hybridoma produced antibody which recognized a dengue-2 virus type-specific determinant and exhibited high titered neutralization but had a low titer by hemagglutination inhibition. Four preparations reacted with a type-specific determinant and exhibited hemagglutination inhibition but did not neutralize. Seventeen hybridomas produced antibodies which were broadly cross reactive in all tests. Only two preparations reacted by complement fixation with dengue-2 antigens; both were cross reactive. Immunofluorescence specificity or cross reactivity correlated with neutralization and/or hemagglutination-inhibition. The dengue-2 virus type-specific antibody useful for identification of dengue-2 infected cells by immunofluorescence has been deposited in the Hybridoma Cell Bank of the American Type Culture Collection.

The relative binding sites of dengue serotype-specific, dengue subcomplex-specific, dengue complex-specific, flavivirus subgroup-reactive, and flavivirus group-reactive monoclonal antibody preparations were identified by using competitive antibody binding assays. A dengue complex-specific epitope, capable of mediating infection enhancement, was identified on a 20,000 dalton protein found on intracellular virions. The other epitopes were assigned relative positions on the E glycoprotein by competitive antibody binding. These could be grouped into 3 linkage groups based on the ability of some monoclonal antibodies to block contiguous binding sites. Some antibodies were able to increase or "promote" the binding of antibodies from other linkage groups. These results suggest that a continuum of antigenic reactivities exist on the E glycoprotein of the dengue viruses, and that the conformation of this glycoprotein may be altered after antibody binding.

Type-specific monoclonal antibodies prepared against the four dengue (DEN) virus serotypes were evaluated for their ability to identify low-passage human and mosquito isolates from Jamaica and West Africa by an indirect immunofluorescence assay. Serotyped human isolates from Jamaican dengue fever patients included 12 DEN-1, two DEN-2, and five DEN-4 viruses. Viruses from West Africa included 84 DEN-2 mosquito strains as well as two DEN-1 and one DEN-2 from humans. Results obtained using the immunofluorescence assay were consistent with virus identifications obtained using the more classical but costly and time-consuming plaque-reduction neutralization test. More viral isolates and higher virus yields were obtained using the C6/36 clone of Aedes albopictus cells rather than LLC-MK2 (monkey kidney) cells. Dengue type-specific monoclonal antibodies detected prototype viral antigens 24-48 hours postinfection in C6/36 cells. This is the first time that monoclonal antibodies have been used to serotype low-passage flavivirus isolates.

SummaryIn 1956 and 1957, 98 to 100% of pigs on farms near Sagiyama north of Tokyo and in mosquito traps at Sagiyama, Tokyo, Zama and Shinhama became infected by JE virus during August. In 1957 extensive studies failed to detect swine infection between April and August. In two rural areas in 1956, swine became infected 2 to 3 weeks before those in Tokyo. Pigs were identified as a major natural source of JE virus for the vector mosquito, Culex tritaeniorhynchus, near Tokyo on the basis of: a) a high incidence of natural swine infection; b) the occurrence of viremia following natural infection; c) demonstration in the laboratory that viremia lasts up to 4 days11 and occurs in titers adequate to infect colonized C. tritaeniorhynchus and cause them to transmit virus to pigs and herons;5 d) the striking propensity of C. tritaeniorhynchus to bite pigs in nature;12 and e) the large, yearly-replenished populations of susceptible pigs near Tokyo.